► Voltage-gated potassium channels are primary determinants of cellular excitability in the mammalian nervous system. The localization of these channels to distinct cellular compartments influences…
(more)

▼ Voltage-gated potassium channels are primary determinants of cellular excitability in the mammalian nervous system. The localization of these channels to distinct cellular compartments influences components of neuronal function, including resting membrane potential, action potential characteristics and neurotransmitter release. Thus, understanding the mechanistic basis of ionchannel localization can provide fundamental insight into human physiology. The overall goal of this dissertation was to elucidate the regulatory mechanisms governing localization and function of the Kv1.3 voltage-gated potassium channel. The sympathetic branch of the autonomic nervous system innervates many organ systems including the kidneys, heart and blood vessels and was used as a model to study endogenous Kv1.3. We found that postganglionic sympathetic neurons express Kv1.3 and that the channel exhibits a striking pattern of localization to the Golgi apparatus in the soma of these cells. Kv1.3 ionic current was also isolated from the soma of these neurons, indicating the channel is a determinant of the electrophysiological properties of sympathetic neurons. In addition, the specific inhibition of Kv1.3 with margatoxin was found to depolarize neuronal resting membrane potential, decrease the latency to action potential firing and increase nicotinic agonist-induced neurotransmitter release. Collectively, these findings demonstrate that Kv1.3 influences the function of postganglionic sympathetic neurons and led to the hypothesis that regulating channel localization may be a mechanism for modulating the activity of these cells. In this dissertation, we propose that the observed Golgi retention of Kv1.3 may be a trafficking-dependent mechanism of channel regulation. To test this hypothesis, we used HEK293 cells as our model system. Our data show that the degree of Kv1.3 Golgi localization is inversely correlated with the amount of channel at the plasma membrane. In addition, the amplitude of Kv1.3 ionic current measured in cells with low Kv1.3 Golgi localization was significantly greater than the current measured in cells with high Kv1.3 Golgi localization. One mechanism for localizing ion channels to the Golgi apparatus involves the Class I PDZ-binding motif (X-S/T-X-Φ). Deletion of the C-terminal PDZbinding motif of Kv1.3 decreased the intracellular Golgi localization of the channel and increased channel localization at the cell surface. Disrupting this canonical binding motif also increased the amplitude of Kv1.3 ionic current. These findings indicate that regulated subcellular distribution of the channel may be a determinant of Kv1.3 surface expression and function.

Doczi MA. Subcellular Distribution of a Voltage-Gated Potassium Channel: the Effect of Localization on Channel Function. [Doctoral Dissertation]. University of Vermont; 2010. Available from: https://scholarworks.uvm.edu/graddis/69

► Sudden cardiac death is one of the major leading causes of death in the United States, affecting about 300,000 people annually on average. Cardiac arrhythmias…
(more)

▼ Sudden cardiac death is one of the major leading causes of death in the United States, affecting about 300,000 people annually on average. Cardiac arrhythmias and ventricular fibrillation can be triggered, at the cellular level, by the presence of aberrations of the cardiac action potential (AP) known as early afterdepolarizations (EADs). EADs are single or multiple voltage oscillations largely induced by the reactivation of L-type Ca2+ currents (ICa,L) during phase 2 and phase 3 of a cardiac AP. Our recent studies using dynamic clamp techniques have suggested that EADs and their arrhythmogenic consequences can be potently suppressed by subtle reduction the ICa,L current non-inactivating (pedestal) component and/or minimal changes (3-5 mV) in the voltage dependence of activation. Exploiting the modulatory effects of L-type Ca2+ channel (LTCC) auxiliary β2 subunits on the non-inactivating component of ICa,L, we sought to investigate the effects of knocking down Cavβ2 subunit expression levels in rabbit ventricular myocytes in the presence of an oxidative stress known to trigger EADs (H2O2). We hypothesized that reducing the expression level of endogenous Cavβ2 decreases the probability of EAD occurrence in cardiomyocytes exposed to H2O2. Using an adenoviral infection to deliver a short hairpin RNA (shRNA) specific for targeting Cavβ2 that inhibits its gene expression by binding to its mRNA transcripts, our results showed that myocytes expressing less Cavβ2 mRNA exhibited no EADs; whereas, the control myocytes infected with GFP alone as a control group were more susceptible to EAD occurrence in 0.6 mM H2O2. These results suggest that Cavβ2 could be a potential target for gene therapy and could give insights to other therapeutic strategies that could possibly be implemented.

▼ Heterotrimeric G-protein signaling pathways modulate synaptictransmission in response to secreted factors. For example, Go, the mostabundant G protein in the nervous system, negatively regulates neuron activityby acting on potassium channels and calcium channels in mammalian systems,although it is possible that other pathways may exist. In my thesis, I used agenetic approach in the nematode C. elegans to study the neuronal function ofGo. I identified an activated Go mutant, which was then used to screen fordownstream components of Go signaling. Several of the proteins I identified inmy screen regulate neuronal excitability, including activation of the calcium- andvoltage-activated potassium channel SLO-1, and the gap junction componentinnexin UNC-9, and inactivation of the novel ionchannel NCA-1. Further geneticscreens demonstrated that the NCA-1 channel requires accessory subunitsUNC-79 and UNC-80. A functional channel complex can be reconstituted byexpressing NCA-1, UNC-79 and UNC-80 in Xenopus oocytes. The channelforms a sodium-selective, voltage-dependent current. I propose a model in whichGo and Gq converge on the RhoGEF UNC-73B and Go downregulates NCA-1activity by antagonizing Gq activation of this pathway.

► Abstract Photoreceptors convert the energy of light into an electric signal to be processed by the visual system. Photoreceptors of nocturnal insects are adapted for…
(more)

▼ Abstract
Photoreceptors convert the energy of light into an electric signal to be processed by the visual system. Photoreceptors of nocturnal insects are adapted for night vision by sacrificing spatial and temporal resolution for improved sensitivity. While the sensitivity-increasing optical adaptations and the temporal properties of light responses have been studied earlier, the intermediate biophysical mechanisms responsible for shaping the captured light into voltage responses were previously not known in detail in any nocturnal species.
Using electrophysiological tools and computer simulations the photoreceptors of the nocturnal cockroach (Periplaneta americana) were studied by characterising 1) the electrical properties responsible for shaping the light responses, 2) the properties of light responses at different stages of light and dark adaptation and 3) properties of low-intensity light stimuli and how they are processed by the photoreceptors.
The high input resistance and whole-cell capacitance were typical for a nocturnal insect, but the two voltage-dependent potassium conductances were closer to those found in diurnal species. The dominant sustained conductance typically associated with day-light vision activated during simulated light responses whereas the lesser transient conductance previously linked to low-light vision did not. Light responses were persistently slow regardless of the adapting light level and saturated at low intensities, indicating a strong adaptation to vision in dim light. Simulations showed that at such low light levels the physical noise caused by random photons determines the information rate and the biological noise, caused by random latency and amplitude of single photon responses, has only a minor effect. At higher intensities the latency variability degraded the information rates but the amplitude variability did not. Thus, photoreceptors of nocturnal animals can sacrifice phototransduction precision in their natural illumination without compromising their coding performance.
Advisors/Committee Members: Weckström, M. (Matti).

► Background: Pulmonary arterial hypertension (PAH) is a condition with severe morbidity and mortality. It is associated with an increase in incidence of all forms of…
(more)

▼ Background: Pulmonary arterial hypertension (PAH) is a condition with severe morbidity and mortality. It is associated with an increase in incidence of all forms of arrhythmias which further increase morbidity and mortality. The monocrotaline (MCT) model of pulmonary hypertension (PH) in the rat is analogous to PAH in humans and was used to study how PH causes arrhythmias. Methods: A single injection of MCT or a volume matched saline injection (control) was given to the rats on day 0 of the protocol. The hearts of both groups of rats were studied in vivo with echocardiography (echo) and electrocardiogram (ECG). The rat’s condition was monitored and they were electively sacrificed when they showed symptoms or on day 28. Live cardiac tissue was studied using the Langendorff preparation and a right atrial preparation that incorporated the sinoatrial (SA) and atrioventricular (AV) nodes. Molecular biology techniques including reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry were used identify changes in the heart caused by PH. The effects of macitentan, an endothelin receptor antagonist used in the treatment of PAH, on the MCT injected rats was assessed using echo and ECGResults: Echo demonstrated that the MCT treated rats developed severe pulmonary hypertension with a decreased pulmonary artery acceleration time (P<0.005) and an increased pulmonary artery deceleration (P<0.005). The MCT treated rats also developed right ventricular hypertrophy (P<0.05) and dilation (P<0.005). The in vivo ECG demonstrated QT prolongation (P<0.005). Ex vivo functional experiments demonstrated QT prolongation (P<0.005) and prolonged ventricular effective refractory period (P<0.005). AV node dysfunction was also seen in the ex vivo experiments with an increased AV effective refractory period (P<0.05), AV functional refractory period (P<0.05) and incidence of complete heart block (P<0.05). RT-qPCR demonstrated significant changes in the mRNA expression of several ion channels and exchanges, Ca2+ handling proteins and autonomic receptors including a downregulation of HCN4 and CaV1.2 in the AV nodal tissues (P<0.05). Treatment of established pulmonary hypertension led to a reduction in the prolongation of the QT interval caused by MCT administration at day 21 (P<0.05).Conclusions: PH causes arrhythmogenic changes including prolonged repolarisation in the working myocardium and AV node dysfunction. These changes may be caused by dysregulation of ion channels and Ca2+ handling proteins. These ion channels and Ca2+handling proteins may play a key role in both physiological and pathological processes within the AV node.

► Influenza viral infection causes several hospitalizations and claims the lives of many people each year. The threat of epidemic and pandemic are more pressing than…
(more)

▼ Influenza viral infection causes several hospitalizations and claims the lives of many people each year. The threat of epidemic and pandemic are more pressing than ever with newly mutated strains developing every year. Understanding the mechanism of infection of influenza can help identify new potential drug targets and help progress the development of antivirals. Currently there are two classes of FDA approved drugs, neuraminidase inhibitors and M2 ionchannel inhibitors, to combat influenza infection. Unfortunately, viral resistance to M2 ionchannel blockers has caused them to stop being used for treatment. This paper focuses on understanding influenzas ability to mutate and it mechanism of infection to develop new M2 ionchannel blockers.
Advisors/Committee Members: Krieg, Paul A (advisor), Krieg, Paul A. (committeemember), Campos, Samuel K. (committeemember), Ahmad, Nafees (committeemember).

► Based on preliminary reports, an extended series of bolamphiphilic oligoester compounds with structural symmetry were synthesized and then tested using a planar bilayer experiment with…
(more)

▼ Based on preliminary reports, an extended series of bolamphiphilic oligoester compounds with structural symmetry were synthesized and then tested using a planar bilayer experiment with the voltage-clamp technique. The main structures of these compounds are identical, consisting of a mono or tri-aromatic core, two octamethylene chains and two benzoyl headgroups which are all connected through ester linkages. The structural variance was provided by the four differently functionalized benzoyl headgroups. The synthetic methods of three to five steps were mainly adapted from the previously reported method.1 The methods successfully produced eight compounds with overall yields of 20 to 30%.
The voltage-clamp data suggested voltage-dependent behaviors occur at low concentrations while Ohmic behaviors require at high concentrations. The activity at low potentials showed relatively erratic behavior but the channels frequently switched between opening and closing states. The activity at high potential lasted longer as the channel maintained a longer state of opening.
The exponential voltage-dependent behaviors were observed at higher potential while the voltage-independent Ohmic behaviors occur at low potential. These channel behaviors are highly time-dependent as there is no control over the stability and the aggregation level for the compounds forming active channels in the membrane. In some cases the current-voltage responses appear to be asymmetrical between the positive and the negative potentials. Mechanisms consistent with the observations are proposed.
Advisors/Committee Members: Fyles, Thomas M. (supervisor).

Previous studies on the rat P2X2 receptor demonstrated that structural modifications to amino acid side chains within the second transmembrane domain lead to receptor activation…
(more)

▼

Previous studies on the rat P2X2 receptor
demonstrated that structural modifications to amino acid side
chains within the second transmembrane domain lead to receptor
activation in the absence of exogenously applied ATP (Rassendren et
al, 1997. Cao et al, 2007. Cao et al, 2009). Present work has been
aimed towards the characterization of these apparently
ATP-independent currents, to investigate the molecular mechanism
underlying this phenomenon using a combination of computer
modeling, amino acid substitution, heterologous expression in
HEK293 cells, the real time modification of engineered cysteines by
MTS compounds and electrophysiological techniques.A screen of
cysteine substituted receptors at TM2 positions (from G323 to T354)
with the membrane permeable MTS compound, MTSP, found that the
compound evoked substantial currents from cells expressing
P2X2[I328C] receptors, but not from cells expressing other TM2
cysteine substituted, nor wild type receptors. MTSP-evoked currents
had similar properties to ATP currents in terms of rectification,
NMDG+ permeability and unitary currents. Further investigation
indicated that hydrophobic, unbranched modifications to the side
chain at position 328 were the most effective.Overall, the results
from this work demonstrate that increasing the length and
hydrophobicity of an unbranched side-chain at position 328 leads to
full receptor activation without the requirement for ATP. These
results suggest that the highly conserved native Ile at position
328 stabilizes the closed of the receptor due to its branched
nature.

Previous studies on the rat P2X2 receptor
demonstrated that structural modifications to amino acid side
chains within the second transmembrane domain lead to receptor
activation in the absence of exogenously applied ATP (Rassendren et
al, 1997. Cao et al, 2007. Cao et al, 2009). Present work has been
aimed towards the characterization of these apparently
ATP-independent currents, to investigate the molecular mechanism
underlying this phenomenon using a combination of computer
modeling, amino acid substitution, heterologous expression in
HEK293 cells, the real time modification of engineered cysteines by
MTS compounds and electrophysiological techniques.A screen of
cysteine substituted receptors at TM2 positions (from G323 to T354)
with the membrane permeable MTS compound, MTSP, found that the
compound evoked substantial currents from cells expressing
P2X2[I328C] receptors, but not from cells expressing other TM2
cysteine substituted, nor wild type receptors. MTSP-evoked currents
had similar properties to ATP currents in terms of rectification,
NMDG+ permeability and unitary currents. Further investigation
indicated that hydrophobic, unbranched modifications to the side
chain at position 328 were the most effective.Overall, the results
from this work demonstrate that increasing the length and
hydrophobicity of an unbranched side-chain at position 328 leads to
full receptor activation without the requirement for ATP. These
results suggest that the highly conserved…

Rothwell, S. (2013). Gating the pore of the P2X2 receptor: the role of
residues within the second transmembrane domain in receptor
activation. (Doctoral Dissertation). University of Manchester. Retrieved from http://www.manchester.ac.uk/escholar/uk-ac-man-scw:202153

Chicago Manual of Style (16th Edition):

Rothwell, Simon. “Gating the pore of the P2X2 receptor: the role of
residues within the second transmembrane domain in receptor
activation.” 2013. Doctoral Dissertation, University of Manchester. Accessed January 21, 2019.
http://www.manchester.ac.uk/escholar/uk-ac-man-scw:202153.

MLA Handbook (7th Edition):

Rothwell, Simon. “Gating the pore of the P2X2 receptor: the role of
residues within the second transmembrane domain in receptor
activation.” 2013. Web. 21 Jan 2019.

Vancouver:

Rothwell S. Gating the pore of the P2X2 receptor: the role of
residues within the second transmembrane domain in receptor
activation. [Internet] [Doctoral dissertation]. University of Manchester; 2013. [cited 2019 Jan 21].
Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:202153.

Council of Science Editors:

Rothwell S. Gating the pore of the P2X2 receptor: the role of
residues within the second transmembrane domain in receptor
activation. [Doctoral Dissertation]. University of Manchester; 2013. Available from: http://www.manchester.ac.uk/escholar/uk-ac-man-scw:202153

The divalent Ca2+ metal ion acts as a universal second messenger in virtually all eukaryotic cells from fungi to plants to mammals. In mammals, Ca2+…
(more)

▼

The divalent Ca2+ metal ion acts as a universal second messenger in virtually all eukaryotic cells from fungi to plants to mammals. In mammals, Ca2+ signaling is vital to a variety of physiological processes including fertilization, cell proliferation, secretion, and muscular contraction. In electrochemically non-excitable tissues, the release of Ca2+ from intracellular stores such as the endoplasmic reticulum is tightly regulated by the inositol 1,4,5-trisphosphate receptor (IP3R). The IP3R Ca2+ release channel is activated by the binding of the small molecule inositol 1,4,5-trisphosphate (IP3) in response to extracellular stimuli such as hormones, growth factors, and neurotransmitters. The conformational changes accompanying IP3 binding were investigated using a biophysical approach. A specific focus of this work is to decipher how signals of ligand binding are transmitted from the N-terminal IP3-binding core to the C-terminal channel domain. To such end, biophysical studies of the ligand-induced conformational changes within the N-terminal domain of IP3R (a.a. 1 – 604) were performed. The results implicated the presence of two flexible linkers which join stably folded domains. This prompted the proposal of a model in which an equilibrium mixture of conformational substrates containing compact and more extended structures co-exist. Determinants within the N- and C-terminal regions of IP3R have previously been reported to be critical to channel function. Employing nuclear magnetic resonance (NMR) as well as biochemical methods, an intermolecular interaction between the S4-S5 linker, the cytoplasmic loop between the fourth and fifth transmembrane helices of IP3R, and the suppressor domain was identified. The determination of the crystal structure of the suppressor domain from isoform type 3 IP3R (IP3R3SUP) allowed us to map the residues involved in this interaction to one face of the molecule. The characterization of this interaction provides insight into the N- and C-terminal determinants essential to the IP3R channel gating mechanism.

► Fluoride ion channels of the Fluc family evolved to combat toxicity from intracellular accumulation of environmental fluoride in microorganisms. Fluc channels are built as antiparallel…
(more)

▼ Fluoride ion channels of the Fluc family evolved to combat toxicity from intracellular accumulation of environmental fluoride in microorganisms. Fluc channels are built as antiparallel dimers with two individual pores running along side of each other and are extremely selective for fluoride over chloride. Although crystal structures are known, the densely packed pore region has precluded identification of the actual ion permeation pathway. With a combination of functional and structural studies, I chart out the span of the Fluc pore and characterize the biochemical requirements for F- transport. A ladder of hydrogen-bond donating residues creates a “polar track” for F- transport pathway. Surprisingly, though the polarity of this track is well-conserved among different homologues, polarity is functionally dispensable at several positions. The positions for which polarity is required are finely-tuned and brook no substitutions. A threonine at one end of the track appears to function through its β-branched methyl group rather than being a hydrogen bond donor. Two essential phenylalanines, each coordinates a F- through an edge-on fashion, presenting an unprecedented aromatic-halide coordination motif. Aromatic, polar and non-polar side-chains all fail to replace the two phenylalanines, but methionine substitution at one position generates a fully functional channel. A Crystal structure of this mutant revealed that methionine side-chain takes a twisted conformation and contacts F- through its partially positive γ-methylene in mimicry of phenylalanine’s quadrupolar interaction. Taken together, these results map out the ion permeation pathway of Fluc and demonstrate the unusual biochemical requirements for selectively transporting the strongly hydrogen bonding fluoride.

▼ PHARMACOLOGY
Pharmacological and Genetic Rescue of Idiopathic Epilepsies
Lyndsey Leigh Anderson
Dissertation under the direction of Professor Alfred George, Jr.
Epilepsy is a common neurological disorder affecting approximately 1% of the population worldwide. Many epilepsy patients achieve complete seizure control with current antiepileptic drugs; however, these medications fail to control seizures in 30% of patients, highlighting the need for novel treatments and for research into the underlying molecular mechanisms of epilepsy. Mutations within voltage-gated sodium channels have been identified in association with epilepsy and several mouse models have been generated to understand how these disease-associated mutations manifest in epilepsy development. The genetically engineered mouse line, Scn2aQ54, expresses a transgene encoding an inactivation-impaired neuronal Nav1.2 channel. Mice expressing the Scn2aQ54 transgene exhibit a severe epilepsy phenotype correlated with increased persistent sodium current in hippocampal neurons. We investigated the antiepileptic potential of preferential persistent sodium current inhibition using ranolazine and the novel compound, GS967. We observed that both ranolazine and GS967 reduced seizure frequency in Scn2aQ54 mice, and GS967 inhibited spontaneous action potential firing in neurons isolated from Scn2aQ54 mice. GS967 was also effective at protecting against seizures in the maximal electroshock model. Additionally, we found that long-term treatment with GS967 improved survival, prevented neuron loss and suppressed mossy fiber sprouting in Scn2aQ54 mice. Heterozygous Scn1a knockout (Scn1a+/-) mice recapitulate the phenotype of Dravet syndrome such as spontaneous seizures and premature lethality. Electrophysiological studies in dissociated hippocampal neurons from Scn1a+/- mice have shown a reduced sodium current density and impaired excitability in GABAergic interneurons suggesting that impaired GABA-mediated inhibition underlies the pathophysiology of Dravet syndrome. We generated a mouse line in which SCN1A is selectively expressed in GABAergic interneurons to directly test the hypothesis that the epilepsy phenotype in Scn1a+/- mice can be rescued by restoring SCN1A in GABAergic interneurons. Utilizing this genetic approach, we were not able to attenuate the reduced lifespan of Scn1a+/- mice by restoring SCN1A selectively in interneurons, suggesting that additional mechanisms may contribute to the reduced survival of Dravet syndrome. However, pharmacological intervention with GS967 was able to successfully rescue the lifespan of Scn1a+/- mice. These results suggest that GS967 is an effective novel antiepileptic drug that can be utilized to probe the pathophysiology of epilepsy.
Advisors/Committee Members: Gregg Stanwood (committee member), Alfred George (committee member), Vsevolod Gurevich (chair), Robert Macdonald (committee member), Jennifer Kearney (committee member).

► This thesis describes the development of an ionchannel system exhibiting dissipative assembly characteristics. In this system an active transporter based on an oligoester fragment…
(more)

▼ This thesis describes the development of an ionchannel system exhibiting dissipative assembly characteristics. In this system an active transporter based on an oligoester fragment terminated in a thioester of 6-aminohexanoic acid (HO2C-Hex-Adip-OctS-Hex-NH2) undergoes thioester cleavage to form a thiol terminated oligoester (HO2C-Hex-ADip-Oct-SH). This fragment was expected to be inactive for ion transport but previous work showed high activity in planar bilayer experiments. In this work, the high activity was shown to be due to the oxidized form of the thiol, the disulfide HO2C-ADip-Oct-SS-Oct-ADip-Hex-CO2H. Air oxidation was found to be quite rapid for the thiol based on ESI-MS (negative ion) and HPLC analysis. Under anaerobic conditions, the thiol fragment was an inactive species for ion transport. In situ air oxidation initiated transport activity associated with the disulfide.
The transporter HO2C-Hex-Adip-Oct-Hex-NH2 was active in planar bilayer experiments and was compared to the disulfide via activity grids. The activity of these two compounds was shown to be distinct from each other by conductance and channel duration differences. The activity of HO2C-Hex-Adip-Oct-Hex-NH2 was shown to die off in a period of 30 minutes at pH 8.2. Techniques were developed to stimulate and monitor activity and bilayer quality so that an inactive condition could be confirmed. The addition of Pr-S-Hex-NH3+-Cl as a fuel was shown to extend the activity of HO2C-Hex-Adip-Oct-Hex-NH2 by eight-fold in 1M CsCl electrolyte. Previous work had established the capability of thioester exchange reactions by a reaction between Pr-S-Hex-NH3+-Cl and benzyl thiol in a homogenous solution. The extended activity indicated that the same process may occur in a heterogeneous bilayer system. An inactive system created by the die-off in activity of HO2C-Hex-Adip-Oct-S-Hex-NH2 was treated with Pr-S-Hex-NH3+-Cl to regenerate activity. This cycle could be repeated once the activity died off again. All these findings are consistent with the dissipative assembly of a membrane transport system.
Advisors/Committee Members: Fyles, Thomas M. (supervisor).

► Phosphatidylinositol bisphosphate (PIP2) directly regulates electrophysiological activity in a diverse family of ion channels whether the effect is stimulatory or inhibitory. Much has been unveiled…
(more)

▼ Phosphatidylinositol bisphosphate (PIP2) directly regulates electrophysiological activity in a diverse family of ion channels whether the effect is stimulatory or inhibitory. Much has been unveiled about the apparent affinity and modulatory function of PIP2 using a chemically modified dioctanoyl PIP2 (diC8), a membrane delimited cytosolic co-factor in inside-out macropatch experiments. Yet, the scarcity of molecular tools that permit fine external control in whole-cell systems has precluded future studies from probing the physiological role of PIP2 in cells in the presence of a fully intact cytoplasm. Here we introduce light as an external control for PIP2 through photocaging of diC8, and test its activation of Kir2.3 (IRK3), an inwardly rectifying ionchannel that has previously shown to possess moderate binding affinity to PIP2, in excised, inside-out macropatches. Our experiments revealed that photocaged-diC8 and irradiated photocaged-diC8 have significantly different activation kinetics than the fully active diC8. Surprisingly, the activation of caged-diC8 by UV irradiation attenuated Kir2.3 activity, while the inactivated diC8 (caged-diC8) resulted in similar magnitude of channel activity compared to the currents elicited by unmodified diC8. Interestingly, we also show that application of both activated (irradiated) and inactive (caged) diC8 in macropatches generated highly fluctuating ionchannel activity.
Advisors/Committee Members: Diomedes Logothetis.

▼ Haemonchus contortus is a parasitic nematode infecting ruminants causing anemia and poor health due to the parasite???s blood-feeding nature. The ability to manage infection has been confounded by the rapid development of antiparasitc drug resistance in H. contortus populations. The Cys-loop superfamily of ligand gated ion channels are well recognized as critical drug targets for many invertebrate specific compounds. With the rise in resistance seen worldwide to existing anthelmintics, novel drug targets must be identified so new treatments can be developed. This thesis describes the identification of three acetylcholine-gated chloride channelss (hco-acc-1, hco-acc-2, and hco-acc-4) genes from the nematode parasite Haemonchus contortus and provides evidence suggesting that they could be developed as a future anthelmintic targets. While these genes appear similar in sequence to the previously characterized C. elegans acc-1, acc-2 and acc-4 genes, Hco-ACC-2 is about 2-fold less sensitive to acetylcholine, perhaps indicating a different in vivo function within the parasite. Further pharmacological analysis of ACC-2 via two-electrode voltage-clamp electrophysiology demonstrated activity of several agonists including carbachol, which elicited a similar response as acetylcholine. Furthermore, in silico protein modelling and agonist docking of all three channels has revealed a unique agonist binding site with several novel residues that appear to be important for binding to cholinergic agonists. Antibodies were generated against the Hco-ACC-1 protein for use in immunolocaliztion studies. Hco-ACC-1 localizes to the anterior half of the pharynx, specifically in pharyngeal muscle tissue in H. contortus.
Advisors/Committee Members: Forrester, Sean.

Callanan, M. (2017). Isolation and partial characterization of three acetylcholine-gated chloride channels in Haemonchus contortus. (Thesis). University of Ontario Institute of Technology. Retrieved from http://hdl.handle.net/10155/850

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Callanan M. Isolation and partial characterization of three acetylcholine-gated chloride channels in Haemonchus contortus. [Thesis]. University of Ontario Institute of Technology; 2017. Available from: http://hdl.handle.net/10155/850

Note: this citation may be lacking information needed for this citation format:Not specified: Masters Thesis or Doctoral Dissertation

▼ Subcellularly resolved, excitable changes (i.e., those induced by electrical or chemical stimuli) in membrane capacitance, influenced by factors including integralmembrane protein activity, lipid densities and membrane-bound water content, may be used to elucidate nonconductive ion-channel conformational state changes, lipid-raft locations and drug–membrane binding processes. However, membrane capacitance has proven difficult to measure, partially because of bandwidth limitations associated with glass/quartz pipettes used during conventional electrophysiology. To address these challenges, techniques introduced in this thesis integrate the principles of extracellular radio frequency (RF) recording with conventional two-electrode voltage clamp (TEVC) to 1) spatially resolve effective membrane capacitance and 2) monitor excitable changes in effective membrane capacitance. Furthermore, this thesis also introduces a new multielectrode method to approximate electrode–electrolyte interfacial impedance, which might prove useful in electric impedance spectroscopic or electric impedance tomographic applications. Specific contributions include the following: 1) A method that simultaneously estimates double-layer and interelectrode (chamber) impedances, in the linear regime of electrode voltage–current sensitivity, during extracellular electrode-based measurements. This method estimates impedance parameters by applying a nonlinear least-squares regression to measurements between various groups or pairs of a three-electrode system and, unlike previous double-layer approximation methods, can be done without the use of multiple calibration solutions or moveable electrode configurations. 2) A platform capable of visualizing the spatial distribution of membrane capacitance, using extracellular RF electrode recordings, around a single cell. The proof-of-concept for this technique is demonstrated with dielectric maps around polarized Xenopus oocyte membranes. 3) Development and characterization of a platform to enable RF impedancebased measurements around voltage-clamped ShakerB-IR-expressing Xenopus oocytes. Data indicated that the platform was most sensitive to effective changes in oocyte dielectric at 300 kHz and 500 kHz. 4) Temporal characterization of changes in voltage-sensitive RF membrane capacitance associated with ShakerB-IR activation (expressed in Xenopus oocytes) and ShakerB-IR–Cu2+ interactions. Results indicate that extracellular RF-impedance-based measurements can temporally and spatially elucidate changes in excitable cell-membrane capacitance and could supplement conventional electrophysiological techniques to provide a broader understanding of cellular biophysics.

► Transport of ions and molecules in solids is a very important process in many technological applications, for example, in drug delivery, separation processes, and in…
(more)

▼ Transport of ions and molecules in solids is a very important process in many
technological applications, for example, in drug delivery, separation processes, and in
power sources such as ion diffusion in electrodes or in solid electrolytes. Progress in the
understanding of the ionic and molecular transport mechanisms in solids can be used to
substantially increase the performance of devices. In this dissertation we use ab initio
calculations and molecular dynamics simulations to investigate the mechamisn of
transport in solid.
We first analyze molecular transport and storage of H2. Different lightweight
carbon materials have been of great interest for H2 storage. However, pure carbon
materials have low H2 storage capacity at ambient conditions and cannot satisfy current
required storage capacities. Modification of carbon materials that enhance the
interaction between H2 and absorbents and thus improve the physisorption of H2, is
needed for hydrogen storage. In this dissertation, corannulene and alkali metal-doped
corannulene are investigated as candidate materials for hydrogen storage. Molecularalso investigated. Using computational chemistry, we predict enhanced H2 adsorption on
molecular systems with modification and hydrogen uptake can reach DOE target of
6.5wt% at at 294 bar at 273 K, and 309 bar at 300 K.
In the second part of this dissertation, we study the lithium ion transport from a
solid electrolyte phase to a solid electrode phase. Improvement of ionic transport in
solid electrolytes is a key element in the development of the solid lithium ion batteries.
One promising material is dilithium phthalocyanine (Li2Pc), which upon self-assembly
may form conducting channels for fast ion transport. Computational chemistry is
employed to investigate such phenomena: (1) to analyze the crystalline structure of
Li2Pc and formation of conducting channels; (2) to understand the transport of Li ions
inside channels driven by an electric field; (3) to study the continuity of the conducting
channels through interface. The study shows Li2Pc has higher conductivity than PEO as
electrolyte.
Advisors/Committee Members: Balbuena,Perla B (advisor), Cagin, Tahir (committee member), Grunlan, Jaime (committee member), Kuo, Yue (committee member), Sue, Hung-Jue (committee member).

► Introduction: Factors that influence atrioventricular (AV) nodal conduction are complex and not well understood. Multiple studies have been performed to explain the mechanisms responsible for…
(more)

▼ Introduction: Factors that influence atrioventricular (AV) nodal conduction are complex and not well understood. Multiple studies have been performed to explain the mechanisms responsible for AV nodal conduction but the AV node (AVN) remains a "riddle". With ageing there is an increase in the incidence of AV nodal dysfunction leading to AV block. Methodology: I have performed electrophysiological (EP) and immunohistochemistry experiments on male Wistar-Hanover rats aged 3 months (equivalent to 20 year old humans; n=24) and 2 years (equivalent to 70 year old humans; n=15). AH interval, Wenkebach cycle length (WCL) and AV node effective refractory period (AVNERP) were measured. I used cesium (Cs+ = 2 mM) to block HCN channels responsible for the funny current "If " (and therefore the membrane clock), and ryanodine (2 μM) to block RyR2 channels responsible for Ca2+ release from the sarcoplasmic reticulum (and therefore the Ca2+ clock) in the two age groups. Protein expression in each group (from n=9 young and n=8 old rats) from different regions of the AV conduction axis: inferior nodal extension (INE), compact node (CN), proximal penetrating bundle (PPB) and distal penetrating or His bundle (His) were studied using immunofluorescence and confocal microscopy. The expression of the gap junction channels Cx43 and Cx40 and ion channel’s including HCN4 (responsible for If current), Nav1.5 (major cardiac Na+ channel responsible for INa) and Cav1.3 (L-type Ca2+ channel), and calcium handling proteins, RyR2 and SERCA 2a (involved in Ca2+ release and reuptake from cardiac sarcoplasmic reticulum, SR) were studied. Semi-quantitative signal intensity of these channels was measured using Volocity software. Structural characteristics of the tissue were studied using histology (Masson’s trichome stain and picrosirius red stain for collagen). Statistical analysis was performed with Prism 6.0. Electrophysiological measurements were performed using Spike2.Results: Without drugs to block the If current and Ca2+ release from the SR, there was a significant prolongation of the AH interval (P<0.005), WCL (P<0.005) and AVNERP (P<0.001) with ageing. In young rats (but not old rats), Cs+ prolonged the AH interval (P<0.001), WCL (P<0.01) and AVNERP (P<0.01). Ryanodine prolonged the AH interval (P<0.01) and WCL (P<0.01) in young and old rats. Immunofluorescence revealed that with ageing: Cx43 is downregulated in the PPB and His (P<0.05); Cx40 is upregulated in the INE and CN (P<0.05); HCN4 is downregulated in the His bundle (P=0.05); Nav1.5 is downregulated in the CN and PB (P<0.05); RyR2 is downregulated in the CN and PPB (P<0.05); SERCA2a and Cav1.3 is upregulated in the PPB (P<0.05). Histology confirmed that with ageing that the cells of CN, PPB and His are more loosely packed and irregularly arranged. There is cellular hypertrophy, decrease in the number of nuclei and increase in the collagen content with ageing. The clinical study has shown that elderly patients with syncope and cardiac conduction system disease are at risk of high…

► Abstract In dim light, reliable coding of visual information becomes compromised, unless the sensitivity of the visual system to light is improved by structural and…
(more)

▼ Abstract
In dim light, reliable coding of visual information becomes compromised, unless the sensitivity of the visual system to light is improved by structural and functional adaptations. Thus far, many adaptations for night vision in the compound eyes of nocturnal insects have been described, but little is known about the mechanisms underlying the electrochemical signalling in their photoreceptors.
In this thesis, whole-cell patch-clamp and mathematical modelling are utilised to study basic electrical properties and ionic currents in photoreceptors of two nocturnal insects, the American cockroach Periplaneta americana and the field cricket Gryllus bimaculatus.
Photoreceptors in both species showed large input resistance, membrane capacitance and phototransduction gain (large single photon responses) compared with most studied diurnal insects, providing improved sensitivity to light. The photoreceptors also expressed two voltage-sensitive outward currents: a transient current and a sustained current. The cricket photoreceptor expressed a dominating transient current, which is a typical characteristic for insects adapted for slow vision in dim light. By contrast, in the majority of cockroach photoreceptors the sustained current dominated, which is more common among fast diurnal species. Model simulations indicated that the sustained current is necessary for improved photoreceptor dynamics. Examination of light-induced currents suggested that the functional variability in cockroach photoreceptors is in part derived from variations in the total area of the photosensitive membrane. Recordings of light-induced currents also revealed that the cockroach light-gated channels are only moderately Ca2+-selective and that the polarisation-sensitive photoreceptors of the cricket may utilise phototransduction machinery in some details different from that in regular photoreceptors. Furthermore, the dynamics and information transfer rates of polarisation-sensitive photoreceptors in the cricket were clearly inferior to their regular counterparts, suggesting that they are not necessary for image formation.

► Lung cancer is the leading cause of cancer-related deaths worldwide. Non-Small Cell Lung Cancer (NSCLC) is the most common type of lung cancer and is…
(more)

▼ Lung cancer is the leading cause of cancer-related deaths worldwide. Non-Small Cell Lung Cancer (NSCLC) is the most common type of lung cancer and is associated with a poor 5-year overall survival rate. In recent years, ion channels have emerged as a putative oncogenic target in cancer research. The aberrant expression of ion channels contributes to several hallmarks of cancer that include uncontrolled growth, evading cell death, cell migration/invasion, and induction of angiogenesis in several types of human cancers. Thus, making ion channels as novel molecular targets in the field of cancer.
This thesis aims to identify the unique role of an electrically silent voltage-gated potassium ionchannel (KCNF1, a.k.a Kv5.1) in NSCLC progression. Interestingly, silencing KCNF1 in NSCLC cell lines via small interference RNA (siRNA)-mediated knock down showed: 1) decreased cell proliferation and 2) reduced cell migration/invasion. Furthermore, through in vitro and in vivo assays, KCNF1 was found to be inversely correlated and a downstream target of Wnt7A- a member of Wnt Signaling Pathway and a tumor suppressor in lung cancer. To elucidate whether its effects in NSCLC cell growth operates via K+ ionchannel function, whole cell patch clamp recordings were performed. Interestingly, stable KCNF1 knockdown cells, as well as Wnt7a expressing cells, showed no significant changes in K+ ionchannel currents. Thus, signifying that KCNF1 acts independent of K+ ionchannel function.
In summary, by using several molecular biology, cell biology, and electrophysiological assays, KCNF1 was identified as a tumor promoter in lung cancer, independent of K+ channel activity. Hence, KCNF1 represents a novel target for the development of future therapies in lung cancer.
Advisors/Committee Members: Winn, Robert A (advisor).

► The Epithelial Sodium Channel (ENaC) is a major determinant in fine-tuning the volume and pressure of blood within the cardiovascular system. Understanding the regulation, structural…
(more)

▼ The Epithelial Sodium Channel (ENaC) is a major determinant in fine-tuning the volume and pressure of blood within the cardiovascular system. Understanding the regulation, structural assembly, and arrangement of ENaC is needed. This study describes a method for identifying residues that participate in structural stability interactions between and within subunits as well as those critical to function. A novel yeast screen was used to describe salt-sensitive phenotypes observed in S. cerevisiae that express ENaC and show growth inhibition. Error prone polymerase chain reaction (EP-PCR) was employed to promote random mutagenesis in the extracellular loop of alpha-ENaC. The levels of growth inhibition were monitored in the yeast strain S1InsE4A and subsequently characterized. The location of the point mutations and the corresponding amino acid transitions cause varying degrees of function. This study successfully illustrates a method for inducing mutations in areas of interest in the gene and screening the resulting mutants. We also identify several mutations of the extracellular domain of alpha ENaC which are critical to function.
Advisors/Committee Members: Booth, Rachell E. (advisor), David, Wendi (committee member), Watkins, Linette (committee member).

► Each year, sudden cardiac death (SCD) attributed to ventricular fibrillation (VF) kills approximately 200,000 people in the United States. However, the mechanisms responsible for VF,…
(more)

▼ Each year, sudden cardiac death (SCD) attributed to ventricular fibrillation (VF) kills approximately 200,000 people in the United States. However, the mechanisms responsible for VF, and therefore VF-related SCD, are incompletely understood. My PhD studies focused on two major topics directly related to the mechanisms of reentry in VF. My general approach was based on the use of neonatal cardiac cell monolayers, gene transfer, immunolocalization, patch clamping and optical mapping techniques. First, I examined how a delayed rectifier potassium channel gene (hERG) involved in cardiac repolarization affects reentry frequency in a ventricular myocyte monolayer model of reentry. The results provided strong evidence for a role of hERG in controlling the frequency and stability of reentry. The mechanisms underlying the acceleration in reentry frequency were shown to be action potential duration (APD) shortening and a transient hyperpolarization after each action potential. APD shortening reduced reentry wavelength which prevented wave front-wave tail interactions and increased reentry stability. The transient hyperpolarization enhanced sodium channel availability and excitability of tissue ahead of the propagating electrical wave front. Together they set the stage for fast and stable reentry that maintains VF. Second, I examined the principle of whether rescuing normal electrical impulse propagation in damaged or fibrotic myocardium using cell therapy would be an effective approach to alter reentry behavior. Electrically excitable cardiac fibroblasts were generated using viral constructs encoding Kir2.1, NaV1.5 and Cx43 proteins. Excitable fibroblasts were able to form monolayers and conduct electrical waves at high velocity. When used to replace normal fibroblasts in heterocellular monolayers, they significantly increased conduction velocity to values similar to those of pure myocytes monolayers. Moreover, during reentry, propagation was faster and more organized, with a significantly lower number of wavebreaks. Altogether, the work accomplished in my dissertation should lead to a better understanding of VF and to the development of novel therapeutic approaches for the prevention of SCD.
Advisors/Committee Members: Jalife, Jose (committee member), Berenfeld, Omer (committee member), Anumonwo, Justus Mukolu (committee member), Lopatin, Anatoli (committee member), Michele, Daniel E. (committee member).

► Cyclic nucleotides-gated (CNG) channels play an essential role in the visual and olfactory sensory systems and are ubiquitously expressed in a variety of neuronal and…
(more)

▼ Cyclic nucleotides-gated (CNG) channels play an essential role in the visual and olfactory sensory systems and are ubiquitously expressed in a variety of neuronal and non neuronal cells. Details of their underlying ion selectivity properties are still not fully understood and a matter of debate in the absence of high resolution structures. Presented in this study are high resolution (1.58-1.95Å) crystal structures and functional analyses of engineered mimics of CNG channels by duplicating their selectivity filter sequences in the background of the bacterial non-selective NaK channel. Mimics share several striking functional similarities in ion selectivity with eukaryotic CNG channels: they are non-selective and permeate Na+ and K+ equally well; externally added Ca2+ serves as a permeating blocker, with the conserved acidic residue in the filter mediating Ca2+ binding. Structures reveal a hitherto unseen selectivity filter architecture that suggests that CNG channel selectivity filters likely comprise three contiguous ion binding sites. The high resolution structures also allow for a thorough characterization of monovalent and divalent ion permeation which, in combination with electrophysiological recordings, offers structural insight into CNG channel function at an unprecedented level of detail.
Advisors/Committee Members: Jiang, Youxing.

Developing central synapses exhibit robust plasticity and undergo marked experience-dependent remodeling. This is particularly evident in sensory systems such as auditory brainstem circuits where synapses mature rapidly to achieve high-fidelity neurotransmission. This depends on a developmental switch in AMPAR composition from slow-gating GluA1 dominant too fast-gating GluA4 dominant known to occur within the first 2 weeks of postnatal development, but the mechanisms underlying this switch remain unknown.
We hypothesize that patterned stimuli mimicking spontaneous or sound evoked activity drives the GluA gating switch. We examined activity-dependent changes in evoked and miniature excitatory postsynaptic currents (eEPSCs and mEPSCs) at the calyx of Held synapse by rupturing the postsynaptic membrane at different time points following 2 minutes of theta burst stimulation (TBS) to afferents in mouse brainstem slices. We found the decay time course of eEPSCs accelerated, manifested as decreases in fast and slow time constants (τf and τs, by exponential fits), but these changes were not apparent until >30 min after TBS. Histogram analyses of the decay time course of mEPSCs for naive and tetanized synapses revealed two τ populations being 0.4 and 0.8 ms, respectively, but the relative weight of the τ0.4 population over the τ0.7 population increased significantly in tetanized synapses.
Such changes are blocked by NMDAR or mGluR1/5 antagonists or inhibitors of CaMKII, PKC and protein synthesis, and more importantly precluded in GluA4-/- synapses, suggesting GluA4 is the substrate underlying the acceleration. Interference of an interaction between the immediate early gene product, Neuronal Activity Regulated Pentraxin (Narp), and AMPARs, blocks this switch, substantiating a role for Narp in excitatory synapse remodeling. These results demonstrate a novel form of plasticity working through coincident NMDAR and mGluR activation to trigger a gating switch of synaptic AMPARs with a temporally delayed expression onset, ultimately enhancing development of high-fidelity neurotransmission at an ultra-fast synapse.

► A number of studies have demonstrated the ability of free fatty acids to activate taste cells and elicit behavioral responses consistent with there being a…
(more)

▼ A number of studies have demonstrated the ability of free fatty acids to activate taste cells and elicit behavioral responses consistent with there being a taste of fat. Here I show for the first time that long chain unsaturated free fatty acid, linoleic acid, depolarizes taste cells and elicits a robust intracellular calcium rise via the activation of transient receptor potential channel type M5. The linoleic acid-induced responses depend on G protein-phospholipase C pathway indicative of the involvement of G protein-coupled receptors in the transduction of fatty acids. Mice lacking transient receptor potential channel type M5 exhibit no preference for and show reduced sensitivity to linoleic acid. Together, these studies show that transient receptor potential channel type M5 plays an essential role in fatty acid transduction and suggest that fat may reflect a bona fide sixth primary taste. Studies to identify the types of taste cells that respond to fatty acids show that both type II and type III taste cells express fatty acid-activated receptors. Fatty acids elicit robust intracellular calcium rise primarily in type II taste cells and a subset of type III taste cells. However, a significant subset of type II taste cells respond to high potassium chloride, which has been broadly used as the indicator for type III taste cells as well, suggesting the expression of voltage-gated calcium channels in these cells. This finding conflicts with previous studies that type II taste cells lack voltage-gated calcium channels. To explore if voltage-gated calcium channels are expressed in subsets of type II taste cells, transgenic mice with type II or III taste cells marked by green fluorescent proteins are used. Results show that a subset of type II taste cells exhibit voltage-gated calcium currents, verifying the expression of voltage-gated calcium channels in these cells. These results question the utility of being able to use high potassium chloride solution to identify unequivocally type III taste cells within the taste buds. A model for the transduction of fatty acids in taste cells consistent with these findings and our previous data is presented.
Advisors/Committee Members: Timothy A. Gilbertson.

▼ Voltage-gated K+ channels are important regulators of
neuronal excitability. Bilaterians have eight functionally distinct
Voltage-gated K+ channel subfamilies: Shaker, Shab, Shaw, Shal,
KCNQ, Eag, Erg and Elk. These subfamilies are defined by sequence
conservation, functional properties as well as subfamily-specific
assembly. Genome searches revealed metazoan-specificity of these
gene families and the presence of prototypic voltage-gated K+
channels in a common ancestor of ctenophores (comb jellies) and
parahoxozoans (bilaterians, cnidarians and placozoans).
Establishment of the gene subfamilies, however happened later in a
parahoxozoan ancestor. Analysis of voltage-gated K+ channels in a
cnidarians species Nematostella vectensis (sea anemone) unveiled
conservation in functional properties with bilaterian homologs.
Phosphoinositide (most notably PIP2) regulation of ion channels is
universal in eukaryotes. PIP2 modulates Shaker, KCNQ and Erg
channels in distinct manners, while PIP2 regulation of Elk channels
has not been reported. I showed that PIP2 modulates the human Elk1
channel in a bimodal fashion: It inhibits voltage activation but
also enhances open state stabilization through mode shift.
Mutational analysis and structural modeling localized three basic
residues on the N-terminal cap domain, S4-5 linker and S6 terminal
as putative binding residues for PIP2. Comparison with Shaker and
HCN channels hints at a common mechanism by which PIP2 modulates
voltage gating in channels.

►Channel replacement therapy represents a new treatment modality that could augment existing therapies against cystic fibrosis. It is based on designing synthetic channel-forming peptides (CFPs)…
(more)

▼Channel replacement therapy represents a new treatment
modality that could augment existing therapies against cystic
fibrosis. It is based on designing synthetic channel-forming
peptides (CFPs) with desirable selectivity, high ion transport
rates and overall ability to supersede defective endogenous
chloride channels. We derived synthetic CFPs from a peptide
initially reconstituted from the second transmembrane segment of
the α-subunit of Glycine receptor (M2GlyR). Our best candidate
peptide NK4-M2GlyR T19R, S22W (p22-T19R, S22W) is soluble in
aqueous solutions, has the ability to deliver itself to the
epithelial cell membranes without the use of a delivery system, is
non-immunogenic, but when assembled into a pore, lacks the
structural properties for anion selectivity. Previous findings
suggested that threonine residues at positions 13, 17 and 20 line
the pore of assembled p22-T19R, S22W and recent studies indicated
that an introduction of positively charged 2, 3-diaminopropionic
acid (Dap) at either T13 or T17 in the sequence increases
transepithelial ion transport rates across the apical membranes of
Madin-Darby canine kidney (MDCK) epithelial cells. This study
focused on further structural modifications of the pore-lining
interface of p22-T19R, S22W assembled pore. It was hypothesized
that singly, doubly or triply introduced Dap residues modify the
pore geometry and that their positively charged side chains impact
discrimination for anions. Dap-substituted p22-T19R, S22W peptides
retain the α-helical secondary structure characteristic for their
parent p22-T19R, S22W. The sequences containing multiple
Dap-substituted residues induce higher short circuit current across
the epithelial MDCK cells compared to peptides with single
Dap-substitutions or no Dap-substitutions. Whole-cell voltage clamp
recordings using Xenopus oocytes indicate that Dap-substituted
peptide assemblies induce higher levels of voltage-dependent but
non-selective ion current relative to p22-T19R, S22W. Studies using
the D-enantiomer of p22-T19R, S22W and shorter truncated sequences
of a full length L-p22-T19R, S22W and L-Dap-substituted peptides
provided evidence that peptide-induced ion transport rates can be
attributed to formation of de novo pathways. Results of preliminary
computer modeling studies indicate that Dap residues affect the
pore geometry but not ion selectivity. Future studies focusing on
modifying the existing electrostatic environment towards anion
selectivity will focus on staggering the charged residues of Dap at
various locations inside synthetic pores.
Advisors/Committee Members: John M. Tomich.

► Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast…
(more)

▼ Glycine receptors (GlyRs) are ligand-gated ion channels (LGICs) that, along with other members of the cys-loop superfamily of receptors, mediate a considerable portion of fast neurotransmission in the central nervous system (CNS). GlyRs are pentameric channels, organized quasi-symmetrically around an ion-conducting pore. Opening of the integral ion pore depends on ligand binding and transduction of this binding signal to the channel gate.
Research presented in this dissertation describes a number of critical electrostatic interactions that play a role in conserving the closed-state stability of the receptor in the absence of ligand, ensuring that receptor activation occurs only upon neurotransmitter binding. These amino acids, aspartic acid at position 97 (D97), lysine 116 (K116), arginine 119 (R119) and arginine R131 (R131) are charged residues that interact with one another through electrostatic attraction. When D97 is replaced with any other amino acid this destabilizes the closed state of the channel and causes spontaneous GlyR channel opening. I show that restoration of this electrostatic interaction in GlyR bearing double mutations in which the charges are swapped (D97R/R119E and D97R/R131D) markedly decreases this spontaneous current. In addition, I investigate how these residues that interact at the interfaces between receptor subunits affect the efficacies of GlyR partial agonists. My work shows that the partial agonist taurine is converted into a full agonist at both D97R and R131D receptors.
Furthermore, I analyzed the structure of the more extracellular part of the transmembrane (TM) 2 segment that lines the ionchannel pore, showing that it is unlikely that this fragment (stretching from T13’ to S18’) is constrained in a true alpha helical conformation. From this work, using disulfide trapping and whole cell electrophysiology, I conclude that a significant level of flexibility characterizes this part of the TM2 domain. This segment includes residue S267, previously shown to be significant for alcohol and anesthetic actions, as well as residue Q266 that, when mutated, produces a hyperekplexia-like phenotype. The range of movement of residues in this region may therefore play an important role not only in channel gating but also in how modulators of GlyR function exert their actions.
Advisors/Committee Members: Mihic, S. John (advisor), Richburg, John H. (advisor), Aldrich, Richard W. (committee member), Duvauchelle, Christine L. (committee member), Pierce-Shimomura, Jonathan T. (committee member).

► Biological and bio-inspired systems using ion transport across a membrane for energy conversion has inspired recent developments in smart materials. The active mechanism in bioderived…
(more)

▼ Biological and bio-inspired systems using ion transport across a membrane for energy conversion has inspired recent developments in smart materials. The active mechanism in bioderived materials is ion transport across an impermeable membrane that converts electrochemical gradients into electrical and mechanical work. In addition to bioderived materials, ion transport phenomenon in electroactive polymers such as ionomeric and conducting polymers produces electromechanical coupling in these materials. Inspired by the similarity in transduction mechanism, this thesis focuses on integrating the ion transport processes in a bioderived material and a conducting polymer for developing novel actuation systems. The integrated membrane has a bilayer lipid membrane (BLM) formed on a conducting polymer, and the proteins reconstituted in the BLM regulate ion transport into the conducting polymer. The properties of the polymer layer in the integrated device are regulated through a control signal applied to the bioderived layer and hence the hybrid membrane resembles an ionic transistor. Due to the bioderived nature of this device, it is referred to as a ‘bioderived ionic transistor’. The research carried out in this thesis will demonstrate the fabrication, characterization and design limitations for fabricating a chemoelectromechanical actuator using the BIT membrane.
The BIT membrane has been fabricated using BLM (DPhPC) reconstituted with protein (alamethicin) to gate Na+ transport into conducting polymer membrane (PPy(DBS)). In this membrane, the bioderived layer is fabricated with proteins by vesicle fusion method and conducting polymer is fabricated by electropolymerization. The bioderived layers, the conducting polymer layers and the hybrid membrane are characterized using electrochemical measurements such as cyclic voltammetry, chronoamperometry, and electrochemical impedance spectroscopy.
The fabrication, characterization and design effort presented in this thesis focuses on the integration of ion transport through the bioderived membrane into volumetric expansion and bending actuation. The characterization efforts are supported by empirical and physics-based models to represent the input-output relationship for both PPy(DBS) actuator and bioderived membrane, and design rules for the proposed actuation platforms are specified. The electropolymerized PPy(DBS) actuator is anticipated to be used in a bicameral device with the chambers kept separated by the DPhPC-alamethicin bioderived membrane. The relationship between the gradient potential, ionic current through the gate, ion concentration, ion transport coefficient in the conducting polymer layer, and the induced tip displacement in the polymer has been concluded from experiments and fitted to the actuation system model. This thesis will also address future directions for this research and anticipated applications for this hybrid actuation concept, such as artificial muscle, drug delivery.
Advisors/Committee Members: Karla Mossi, Vishnu Sundaresan.

An investigation of group I monovalent cation selectivity in biological molecules and macrocycles is presented in this thesis. This ion selectivity is responsible for many…
(more)

▼

An investigation of group I monovalent cation selectivity in biological molecules and macrocycles is presented in this thesis. This ion selectivity is responsible for many important and crucial biological processes such as shaping action potentials in excitable cells and the proper functioning of many enzymes. A number of mechanisms have been proposed as to how this is achieved and it is the intent of this thesis to elucidate and discuss known mechanisms, as well as to identify new ones. Computational techniques are used to undertake this study, allowing the probing of time, space and energies unattainable through traditional means of experiment. A coherent description of the means of generating ion selectivity is presented, leading to an explanation of selectivity in a wide range of molecules, as well as having ramifications for the design of novel ion selective structures. Chapter 1 introduces the history and the current state of the field. Chapter 2 contains a synopsis of the following chapters. Chapter 3 presents and explores the fundamental mechanisms of ion selectivity and their importance in a number of molecules. Chapter 4 expands on this, detailing situations where reality may deviate from these simple situations described in chapter 3.

Chapter 5 elucidates how enforcing specific coordination numbers around ions creates ion selectivity. Chapter 6 introduces a new mechanism of ion selectivity, the reduced ligand fluctuation model, in which the reduction in the thermal motion of ligands in a binding site can yield ion discrimination. We suggest that this may play a role establishing selectivity in ion-coupled amino acid transporters. Chapter 7 investigates the impact of molecular strain on ion selectivity through the study of two bacterial ionophores, valinomycin and nonactin. Chapter 8 presents preliminary work that uses the knowledge gleaned from the previous chapters to aid in the in-silico construction and testing of novel ion selective pores. The final chapter concludes this thesis and proposes avenues for further investigation.

An investigation of group I monovalent cation selectivity in biological molecules and macrocycles is presented in this thesis. This ion selectivity is responsible for many important and crucial biological processes such as shaping action potentials in excitable cells and the proper functioning of many enzymes. A number of mechanisms have been proposed as to how this is achieved and it is the intent of this thesis to elucidate and discuss known mechanisms, as well as to identify new ones. Computational techniques are used to undertake this study, allowing the probing of time, space and energies unattainable through traditional means of experiment. A coherent description of the means of generating ion selectivity is presented, leading to an explanation of selectivity in a wide range of molecules, as well as having ramifications for the design of novel ion selective structures. Chapter 1 introduces the history and the current state of the field. Chapter 2 contains a…

Thomas, M. (2011). A computational study of the origin of group I ion selectivity in biological molecules. (Doctoral Dissertation). University of Western Australia. Retrieved from http://repository.uwa.edu.au:80/R/?func=dbin-jump-full&object_id=33306&local_base=GEN01-INS01

Chicago Manual of Style (16th Edition):

Thomas, Michael. “A computational study of the origin of group I ion selectivity in biological molecules.” 2011. Doctoral Dissertation, University of Western Australia. Accessed January 21, 2019.
http://repository.uwa.edu.au:80/R/?func=dbin-jump-full&object_id=33306&local_base=GEN01-INS01.

Thomas M. A computational study of the origin of group I ion selectivity in biological molecules. [Internet] [Doctoral dissertation]. University of Western Australia; 2011. [cited 2019 Jan 21].
Available from: http://repository.uwa.edu.au:80/R/?func=dbin-jump-full&object_id=33306&local_base=GEN01-INS01.

Council of Science Editors:

Thomas M. A computational study of the origin of group I ion selectivity in biological molecules. [Doctoral Dissertation]. University of Western Australia; 2011. Available from: http://repository.uwa.edu.au:80/R/?func=dbin-jump-full&object_id=33306&local_base=GEN01-INS01

▼ Claudins constitute a family of tight junction
transmembrane proteins whose first extracellular loop (ECL1)
determines the paracellular permeability and ion selectivity in
epithelia. Claudin-2 forms a paracellular cation pore. We are
interested in the molecular mechanism of ion selectivity of
claudin-2 from a structural-functional perspective. In chapter 2,
we explored the role of two highly conserved cysteines in ECL1 by
hypothesizing that these extracellular cysteines are linked by an
intramolecular disulfide bond. We found that the single cysteine
mutants can form a claudin-2 homodimer, suggesting that the two
conserved cysteines normally form an intramolecular disulfide bond
in wild-type claudin-2. We also found that the disulfide bond is
necessary for pore formation. In chapter 3, we tested the role of a
highly conserved aromatic residue near the pore selectivity filter
of claudins by hypothesizing that it contributes to cation
selectivity by cation-pi interaction with the permeating cation.
The Y67L mutant showed reduced cation selectivity compared to
wild-type claudin-2 due to the decreased Na⁺ permeability, without
affecting the Cl⁻ permeability. The Y67A mutant enlarged the pore
size and further decreased the charge selectivity due to an
increase in Cl⁻ permeability. The Y67F mutant restored the Na⁺
permeability, Cl⁻ permeability, and pore-size back to wild-type. We
conclude that the conserved aromatic residue near the cation pore
domain of claudins contributes to cation selectivity by a dual role
of cation-pi interaction and a luminal steric effect. In chapter 4,
we aimed to map out all pore-lining residues of claudin-2 through
comprehensive cysteine-scanning mutagenesis of ECL1. We screened 45
cysteine mutants of the ECL1 in polyclonal MDCK II Tet-off cells
and found nine pore-lining residues. Next, we stably expressed
these candidates in monoclonal MDCK I Tet-off cells for
confirmatory studies. The mutants had similar ion permselectivity
and pore size as wild-type claudin-2. Nevertheless the conductance
inhibition assay of a panel of MTS reagents revealed distinct
patterns of blockage effect and varying kinetics of reaction. In
conclusion, we identified all pore-lining residues of claudin-2
with distinct geometrical location. This can be applied to future
x-ray crystal structures and molecular modeling of claudins to
further understand the molecular mechanism for paracellular ion
transport.
Advisors/Committee Members: Yu, Alan S. L. (Committee Chair), McDonough, Alicia A. (Committee Member), Farley, Robert A. (Committee Member), Langen, Ralf (Committee Member), Peti-Peterdi, Janos (Committee Member).